Interferons are single chain polypeptides secreted by most animal cells in response to a variety of stimuli, including viruses, mitogens and cytokines. Interferons participate in the regulation of cell functions and mediate antiproliferative, antiviral and immunomodulatory effects. Thus, they are of great interest therapeutically. Native interferons are divided into three major types, based on the cell types from which they are primarily derived, namely, interferon-α (from leukocytes), interferon-β (from fibroblasts), interferon-γ (from immune cells). Interferon-β (IFN-β) exhibits various biological and immunological activities and as a result has potential applications in immunotherapy, antitumor, anticancer and antiviral therapies. Numerous investigations and clinical trials have been and are being conducted based on anticancer and antiviral properties of both wild-type and recombinant IFN-β. Clinical trials using recombinant IFN-β in the treatment of multiple sclerosis also have been conducted.
Most cytokines, including native IFN-β, have relatively short circulating half-lives. Consequently, in order for IFN-β to be effective as a therapeutic agent, it must be administered in large and frequent doses to a patient; however, this often leads to toxic side effects. Therefore, it is highly desirable to produce forms of IFN-β that have prolonged circulating half-lives compared to the native cytokine. Furthermore, for production purposes it is useful to produce forms of IFN-β that are easy to express and purify in large amounts.
Human IFN-β (huIFN-β) is a glycoprotein of 166 amino acids and has a four helix-bundle structure. Recombinant huIFN-β may be commonly produced for use as a therapeutic in either a prokaryotic or a mammalian expression system. However, when proteins that are normally secreted, such as huIFN-β, in a mammalian environment are produced in a prokaryote, the effect of prokaryotic expression on protein folding and on potential post-translational modifications needs to be addressed. For example, in mammalian cells, most proteins destined for the extracellular milieu are folded in the oxidizing environment of the endoplasmic reticulum (ER), which promotes the correct formation of disulfide bonds. In contrast, the reducing environment of the prokaryotic cytosol interferes with the formation of cysteine bonds. In addition, proteins expressed in prokaryotic systems lack some post-translational modifications, such as N-linked glycosylation, which likely aid in the correct folding of the protein, increase the stability of the folded protein, and decrease the immunogenicity of the administered protein.
For example, when intact wild-type IFN-β is expressed in a prokaryotic expression system, it does not fold properly and forms aggregates. This can be overcome by mutating the free cysteine at position 17 of the mature IFN-β protein to, for example, a serine. This cysteine at position 17 is not involved in a disulfide bond. See, for example, U.S. Pat. No. 4,737,462. In contrast, when intact wild-type IFN-β is produced in a eukaryotic expression system, where the environment is appropriate for correct folding of the IFN-β protein, improper folding and aggregation are not observed. Because IFN-β protein appears to fold properly and not to aggregate when expressed in a eukaryotic expression system, this suggests that glycosylation plays an important role in proper folding of the IFN-β protein. Recombinant IFN-β produced in a eukaryotic expression system undergoes glycosylation, although it may not have the precise glycosylation pattern of the native IFN-β. See, for example, U.S. Pat. No. 5,795,779. Whereas glycosylation of IFN-β does not seem to be essential for its biological activity, the specific activity of glycosylated IFN-β in bioassays is greater than that of the unglycosylated form. Indeed, IFN-β produced in a eukaryotic expression system, such as a mammalian expression system, is substantially non-aggregated, but does form aggregates when the glycan moiety is removed. Therefore, the glycosylated form of IFN-β is desirable for therapeutic use as its biophysical properties are closer to those of the native protein than the unglycosylated form.
In addition, it has been found that linking a protein of interest “X” to an immunoglobulin Fc domain “Fc” to create an Fc-X fusion protein (“immunofusin”) generally has the effect of increasing protein production significantly. This is believed to occur, in part, because the Fc moiety of the fusion protein, commonly referred to as the expression cassette, is designed for efficient secretion of the fusion protein, and in part because proteins are being produced and secreted from mammalian cells that are normally active for secretion. A further advantage of creating Fc-X fusion proteins is that the resultant immunofusins exhibit an increased circulating half-life as compared to the free proteins of interest, which can be a significant therapeutic advantage.
There is, therefore, a need in the art for biologically active immunofusins including an Fc moiety fused to an IFN-β moiety optimized to have biophysical properties that are close to those of native IFN-β.